Download General Microbiology

By
Prof. Dr. Asem Shehabi and Dr. Suzan Matar
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The Microbial World
 The microbial world is composed of
microbes/ microorganisms
- Free living
- Commensal
- Pathogenic
 Consists of
-
Bacteria
Fungi (Yeast/ Moulds)
Algae
Protozoa/ Parasites and Helminths
Viruses
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 Microbiology is concerned with the study of these
microbes.
- Mostly are beneficial for human life
- Few species cause harmful effects
 Microorganisms are unicellular cell, too small to
be seen with the naked eye, recognized by light
microscope.
 Bacteria, fungi & parasites, size generally above >
0.1 um and < 10um
 Most microbes capable of grow & existence as
single organism or together with others. Widely
distributed in human, animal, nature.
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Microbiology
 Viruses sizes < 0.01um
- Composed of only DNA or RNA.
- Grow only in living cells/tissue culture.
- Can’t be considered true microorganisms.
- Their presence structures can be seen only with
electron microscope.
 Microbiology has many areas of specialization
including Bacteriology, Mycology (fungi), Virology,
Medical microbiology, Immunology, Food microbiology,
Biotechnology, Microbial genetics, Industrial
microbiology, Agriculture Veterinary.
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Bacteria
 Bacteria
- Unicellular
microorganisms.
- Size (0.2 um Diameter,
0.2-10 um Length)
- Having a variety of shapes.
Coccus/cocci,
Bacillus/bacilli or Rods
Coccobacilli
Spiral forms- spirochetes
Vibrios
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- Growth patterns & metabolic characteristics
allowing their classification.
- Individual cells may be arranged in pairs or
clusters or chains. Their morphologies are useful
for the identification & classification of bacterial
Genera and Species.
- Colored by Gram-stain or other stains (Fig-1)
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Figure -2 Bacteria Cell structure
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Bacterial Cell structures-1
 Cell wall structures:
- A rigid cell wall
- Cell wall is the basis for
classification of bacteria
into Gram-positive & Gramnegative by Gram-stain
- Composed of many
peptidoglycan layers
N-acetylglucosamine
N- acetylmuramic acid
Pentapeptide.
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Cell wall Gram-positive bacteria-3
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Cell wall
- Lipopolysaccharide (LPS or
endotoxins) in gram negative
bacteria cause endotoxic shock.
o LPS structures are composed
of lipid A, which binds to the
outer membrane
o Endotoxic portion/Toxicity
associated with Lipid A
molecule, causing toxic shock,
high fever, bacterial sepsis
o The polysaccharide structure
appears on the cell surface,
serving as an antigenic
determinant -O antigeno Host body develops during
bacterial Infection..Anti-O
Antibodies
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Cell Wall Gram-negative bacteria-4
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Gram-Stain
A- Gram-positive Stained purple (crystal violet):
Examples: Staphyloccocus, Streptocooci,
Bacillus
Teichoic acid and Lipoteichoic acid within cell wall is
significant for staining gram positive bacteria
* Protoplasts L-form due to lysozyme effect, loss
most Cell wall, Burst +Lysis
B- Gram-negative Stained red (safranin)
Examples: Enteric bacteria group. E. coli,
Klebsiella, Salmmonella, Pseudomonas
* Spheroplasts (L-form) in gram negative
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Cytoplasmic membrane
- Phospholipid bilayer ..Hydrophilic-Hydrophobic
structures
- Cytoplasmic membrane lacking sterols..Contains
various membrane proteins, enzymes and
permeases/ membrane transport proteins..
- Responsible for transport of ions, nutrients and waste
across the membrane and control the cell plasma
contents
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 Bacterial genome composed of:
- One single supper coiled DNA chromosome,
about 1.5mm ..1000 size of the bacterial cell
- Plasmids (>1), Bacteriophage DNA/RNA
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 Bacterial Flagella:
- Organs of motility,
- Composed of flagellins (polymer
-
-
proteins) long filament
Length up to 20 um
Attachment/Adhesion epithelial
host cells.. Infection
Nutrition..
Single polar flagellum
(monotrichous)
Several polar flagella at one,
each end of the cell or covering
the entire cell surface
(peritrichious)
Antigenic determinants
(H-antigen).. observed during
bacterial infection.
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Bacterial Cell structures-2
 Fibmriae and Pili:
- Small surface appendages (protein)
- Few numbers Pili
(Sex pili in conjugation)
- Large numbers fimbriae
- Both pili and fimbraie have specific
functions such as
* Attachment/Adhesion to
host epithelial cells/colonization
* Antigenic determinants..Anti-F
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 Capsules:
- Surface layer of cell wall
- Slime layer composed
mostly of high molecular
weight polysaccharides
- Functions
• Provide resistance to blood
phagocytosis
• Avoid the killing effects of
host lysosomal enzymes,
Serve as antigenic
determinants (K-antigen)
• Major virulence factor in
certain bacteria
• Biofilm formation on
medical devices
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Other components in bacterial cells
- 70S ribosomes
- Mesosomes
Infoldings in the plasma membrane,these are rich in
enzymes that helps to perform functions like cellular
respiration, DNA and cell division (most important
function)
- Storage granules -Lipids, glycogen, polysaccharides,
sulfar, phosphate and others storage compounds.
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Virulence factor
Any bacterial part/product associated with
pathogenic potential, causing human/animal
infection/disease.
 colonization of specific tissue in the host (this
includes attachment to cells)
 immunoevasion, evasion of the host's immune
response
 Immunosuppresion, inhibition of the host's
immune response
 entry into and exit out of infected host cells
 obtain nutrition from the host cells
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Spore-Forming Bacteria
o ENDOSPORE FORMATION:
sporulation begins when vegetative (actively growing cells)
exhaust their source of nutrients, begin of forming
endospores, Common in nature (Figure 4).
o Spore forming Bacteria are very resistant to lysozyme, heat,
radiation, drying and can remain dormant for hundreds of
years in nature.
o Germinate & return to the vegetative state
o Application of moist heat at100-120oC for a period of 10-20
min may be needed to kill spores.
o Aerobic Bacillus group & Anaerobic Clostridium
- Develop Endospore formation
- Both are widely distributed in nature, intestinal -human and
animals.
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Figure 4
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Bacterial Growth by Binary fission
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Growth & Nutrition-1
 Requirements for bacterial growth: temperature,
oxygen, water, pH, temperature, source of carbon,
nitrogen ( organic compounds), inorganic salts.. Na, K,
S, P, Ca, Mg, Cl, Fe, vitamins, etc.
 Obligate Aerobic bacteria :M. tuberculosis,
P.aeruginosa grow using aerobic respiration by
oxidation.. oxygen serves as the terminal electron
acceptor for electron transport system to metabolise
substances..sugar, fats, proteins..obtain energy.
 Aerobic bacteria utilizing oxygen during their growth by
producing oxidizing enzymes:
 Peroxidase: H2O2  2H2O+NAD.
 Catalase: H2O2  2H2O+O2
 Superoxidase dismutase: O2-  H2O2 +O2
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Growth & Nutrition-2
 Certain Pathogens grow with reduced level of oxygen.
Microaerophilic bacteria such as Neisseria spp.
 Facultative anaerobes.
Grow by presence/absence of oxygen
Most human pathogens , Intestinal flora, Normal body
flora.. E.coli ,Staphylococci, streptococci,
 Obligate Anaerobic
Bacteria grow by absence of oxygen.. use recipient
inorganic molecule..Sulfate, Nitrate.
- Anaerobic respiration / Fermentation..
Mostly found in intestinal tract (95-99%), Mouth
&Vagina(90%). Examples Anaerobes: Gram-ve
Bacteriodes fragillis, G+ve Clostridia.
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Oxygen requirement by bacteria
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Growth & Nutrition-3
 Bacteria classified by the source of their energy/
oxidation-reduction process into two groups:
 Heterotrophs derive energy from breaking down
complex organic compounds.. protein, sugar, fats.
Human tissues.. All commensals & pathogens
 Saprophytic bacteria/ Nonpathogenic obtain energy
by fermentation/respiration. Found in nature and in
decaying material, soil and water, important for
circulation of minerals.
 Autotrophs fix carbon dioxide to make their own
growth source.
- Photoautotrophic using light energy
- Chemoautotrophic using oxidation of nitrogen, sulfur,
other elements. Such as sulfur & nitrogen fixing
bacteria found in the environment.
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Culture Media:
 Nutrients (carbohydrates , Fats & proteins, blood,
body tissue &fluid)
 Minerals
 Water
 pH
 Tempertaure
- Growth cultures (Fig 7):
 Broth medium..Nutrient broth in tube
 Solid medium.. Nutrient agar, MacConkey agar, Blood
agar in culture plates
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Bacterial Growth –MacConkey agar
& Tube Broth (Fig-7)
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 Types of culture media:
1. General culture media: growth of most human
pathogens, Gram-ve & Gram-ve bacteria
Nutrient agar
Blood agar
Chocolate agar
2. Selective & differential media..
MacConkey agar ( Bile salts+ Lactose+neutral red
dye) Inhibits Gram-ve bacteria enhance the growth
of E.coli, other enteric bacteria
3. Other Selective media: S-S agar .. For isolation of
Salmonella, Shigella , V.cholerae from stool
specimens.
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Neutrophilic bacteria
Grow best pH (7-7.2) Most
human-animal commensals &
pathogens
 Acidophilic bacteria
< 5 pH.. Lactobacilli
 Mesophilic bacteria
Grow at (20-40C). Most human
commensal & pathogens.
 Psychrophilic bacteria
(<10C),
 Thermophiles bacteria
(> 60C)..Common in hot spring
water
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Bacterial growth-1
 Bacterial growth is the
division of one bacterial
cell into 2 identical
daughter cells 1,2,4,8,16..
by binary fission.
 Generation time
(15-25 min), most human
commensal & pathogens.
 Each single cell produces
one colony 103 -109 cells
on solid culture plate
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 Baterial Growth Curve:
- 4 phases of visible growth: Lag, Log, Stationary,
death/ decline.
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Measurements of Microbial Growth
 Direct Methods: Count individual cells
(a)
Microscopic
(living and dead cells)
(b)
Viable plate count
(c)
Membrane filtration
 Indirect Methods: Measure effects of microbial growth
(a)
Turbidity
(b)
Metabolic assay
(c)
Dry weight determinations
Enumeration of bacteria is very important in study
research to detect antibiotics .
Standard Plate Count
Serially diluted samples are plated out and bacterial count expressed in CFU/ml.
Turbidimetric
Turbidimetric measurements as indicators of bacterial growth.
The greater the turbidity the larger the population density.